Project summary/Abstract Severe combined immunodeficient (SCID) pigs are a valuable resource in biomedicine. For instance, pigs lacking RAG2 and IL2RG can be a useful model in stem cell transplantation, immunology, and infectious diseases because they lack all major lymphocytes (B-T-NK-). However, application of the models has been limited because the pigs cannot be propagated through natural breeding due to their immunodeficiency; no germ cells (spermatozoa and oocytes) can be produced from these animals. Somatic cell nuclear transfer (i.e. cloning) or bone marrow transplantation may be applied to overcome the shortcoming, but it is labor intensive and inefficient. The embryo complementation (or blastocyst complementation) technique provides an opportunity to exhibit two or more phenotypes in a single animal. The objective of this project is to establish a foundation system that can be applied to produce germ cells from any type of immunodeficient pigs. Our hypothesis is that embryo complementation of two embryo types, each carrying immunodeficient and germ-cell depleted phenotype, will allow animals from the embryos to have a functional immune system while restricting germ-cell development only from the immunodeficient genetic background. We will address our hypothesis by two specific aims. Aim1. Establish chimeric pigs that produce spermatozoa carrying modified RAG2/IL2RG. Aim2. Establish chimeric pigs that produce oocytes carrying modified RAG2/IL2RG. Novel germ-cell depleted pig models, which can expand our understanding of germ cell development, will also be produced and characterized. By introducing targeted modifications during embryogenesis through the use of CRISPR/Cas9 system, we will rapidly generate these pig models within the timeframe of R21 and demonstrate their ability to produce germ cells. Our extensive experience in using the CRISPR/Cas9 system in pigs, especially during embryogenesis, will ensure successful completion of the proposed aims. The knowledge obtained from the project can expand the use of immunodeficient pigs by establishing a strategy to share their germ cells with other institutes and help understand mechanism of germ-cell development using large animal models preventing different stage of germ-cell development. Because pigs are physiologically, anatomically, and immunologically similar to humans, they can be a suitable model in biomedicine as a preclinical translational model. The success of this proposal will diversify animal models that can be applied in a wide array of biomedical research areas such as HIV, X-linked immunodeficiency, cell transplantation therapy, and autoimmune diseases.